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Files in this Data Supplement:
Fig. S1. Experimental manipulation of erythroblasts in mouse embryos. Illustration of the experiments performed to sequester blood cells in the blood islands using polymerized acrylamide and to rescue the effects of lowered effective viscosity by injecting hetastarch into the circulation. Manipulated embryos were either allowed to grow in roller culture for 24 hours or were injected with dextran to measure plasma flow using FRAP. The picture of the embryo included in this figure shows the result of fluorescein-dextran injection into the plexus after acrylamide polymerization (scale bar: 100 μm). GFP+ erythroblasts are evident in the blood islands (punctuate green) and fluorescein-dextran (also green) is filling the plexus channels. Dextran is distributed through the entire plexus in acrylamide-treated embryos.
Fig. S2. Time-lapse of erythroblast expansion from blood islands. A movie that progressively follows a 9-somite Mlc2a−/−; Tg (ε-globin-GFP) embryo over 7 hours with images taken every 6 minutes with a 10× Plan-Neofluar 0.3NA objective lens. The images shown were taken at (A) 0, (B) 1.6, (C) 3.1, (D) 4.6, (E) 6.1 and (F) 7 hours. Erythroblasts enter the field of view within single frames, indicative of the presence of flow, but stop flowing and accumulate to form larger clumps. Oscillations of cells are evident when watching the movie and evidence of oscillations in Mlc2a embryos is shown in Fig. 5. Scale bar: 100 μm.
Movie 1. Time-lapse analysis of the initiation of erythroblast circulation. Images were taken of a 6-somite mouse Tg(ε-globin-GFP) embryo every 6 minutes for a total of 12.1 hours. The yolk sac (YS), heart (H) and somites (S) are indicated in Fig 1. Initially, very few erythroblasts are visible in the yolk sac and embryo (t=0). One hour later, an increased number of erythroblasts appear in the yolk sac circulation. After 8.1 hours, significant numbers of erythroblasts circulate through the yolk sac and embryo proper, and these numbers continue to increase through the end of the movie. Clumps of erythroblasts are seen as some cells stop circulating, whereas others continue to move freely.
Movie 2. Erythroblast motion in Mlc2−/− embryos. The motion of erythroblasts (green) within yolk sac vessels of Mlc2−/− embryos was imaged at two frames/second and demonstrates an oscillatory motion with little bulk forward flow. The movement of individual erythroblasts throughout the cardiac cycle is illustrated in Fig. 5.
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